The major goal of this project is to define the structure of the retinal/pineal gene, N-acetyltransferase (NAT) and to examine NAT gene expression in visual and neural transduction. The primary objective of this five year research proposal is to answer the question: Is the retinal/pineal NAT enzyme a key component of a circadian driving oscillator and is the retinal/pineal NAT enzyme controlled by environmental cues at the molecular level? This project will investigate one specific phase of this endogenous rhythm and the entrainment by the light:dark cycle of the rate=limiting enzyme that synthesizes melatonin. The pineal is an excellent system to study all steps in neurochemical transduction, including ligand-receptor interactions, second messenger regulation, and selective regulation of specific genes. Two characteristics for studying molecular mechanisms of pineal neurotransduction are (1) innervation by sympathetic fibers involving alpha- and beta-receptors; and (2) high amplitude circadian rhythms in biochemical activities, including some that are rapidly responsive to known molecular triggers. Although the retina shares similar proteins and biochemical pathways with the pineal, the role of melatonin in the retina is not understood. The experiments will characterize the retinal/pineal tissue-specific genes coding for NAT. Normal tissue-specific expression will be analyzed in retinas and pineals of rats and inbred mice. Primary pineal cell cultures will also be used to study the role adrenergic regulation has in gene expression. The project will have three specific aims: 1. Isolate full-length cDNAs and characterize cDNA probes encoding the retinal/pineal enzyme, NAT, by screening a rat pineal expression library. 2. Generate antisera for immunological probes against recombinant fusion proteins and synthesized peptides identified from the translated cDNAs encoding NAT. 3. Apply these recombinant and immunological probes to localize, characterize and measure changes in circadian regulation and environmental modulation of their mRNA and protein for NAT. The experiments described will employ immunological and recombinant DNA tools to examine the influence of environmental cues on the genes expressed in the pineal and retina. The pineal and retina have been extensively studied by physiological, pharmacological and biochemical approaches. These studies will extend these studies concerning the fundamental molecular nature of circadian rhythms in the nervous system. Biological circadian rhythms are important not only in our daily lives, but in maintaining our health throughout life. Knowledge of the normal structure of genes expressed in the pineal and retina may lead to a better understanding the involvement of the melatonin synthesizing enzyme in visual and neural transduction.